Halophile Aldehyde Dehydrogenase from Halobacterium salinarum Hyo-jeong Kim, Won-A Joo,† Chang-Won Cho, and Chan-Wha Kim* School of Life Sciences and Biotechnology, Korea University, Seoul, Korea Received August 5, 2005
Abstract: Halobacterium salinarum is a member of the halophilic archaea. In the present study, H. salinarum was cultured at various NaCl concentrations (3.5, 4.3, and 6.0 M NaCl), and its proteome was determined and identificated via proteomics technique. We detected 14 proteins which were significantly down-regulated in 3.5 M and/or 6 M NaCl. Among the identified protein spots, aldehyde dehydrogenase (ALDH) was selected for evaluation with regard to its potential applications in industry. The most effective metabolism function exhibited by ALDH is the oxidation of aldehydes to carboxylic acids. The ALDH gene from H. salinarum (1.5 kb fragment) was amplified by PCR and cloned into the E. coli strain, BL21 (DE3), with the pGEX-KG vector. We subsequently analyzed the enzyme activity of the recombinant ALDH (54 kDa) at a variety of salt concentrations. The purified recombinant ALDH from H. salinarum exhibited the most pronounced activity at 1 M NaCl. Therefore, the ALDH from H. salinarum is a halophilic enzyme, and may prove useful for applications in hypersaline environments. Keywords: H. salinarum • 2-DE • MALDI-TOF • ESI-Q TOF mass spectrometry • ALDH
Introduction Halophilic microorganisms produce a variety of stable enzymes (including many hydrolytic enzymes, such as DNases, lipases, amylases, gelatinases, and proteases) which are capable of functioning under conditions which would lead to the precipitation or denaturation of most other proteins. Novel halophilic biomolecules can also be used in a host of specialized applications, e.g., bacteriorhodopsin for bio-computing, gas vesicles for the bioengineering of floating particles, pigments for food coloring, and compatible solutes for stress protectants.1 Recently, the biotechnological uses of halophilic microorganisms have expanded, and additional possible applications are currently being investigated. As a substantial number of industrial processes and a variety of waste-water treatment schemes involve hypersaline conditions, these peculiar characteristics of halophilic enzymes may hold great promise with regard to industrial applications. Recent advances * To whom correspondence should be addressed. School of Life Sciences and Biotechnology, Korea University, 1, 5-ka, Anam-dong, Sungbuk-ku, Seoul 136-701, Korea. Tel: +82-2-3290-3439. Fax: +82-2-3290-3957. E-mail:
[email protected]. † Current address: Wistar Institute, Philadelphia, USA.
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in both proteomic and genomic technology have recently been applied to research concerning H. salinarum. We have attempted to apply the whole proteome approach to the identification and development of useful enzymes from this organism. We reported in a previous study that proteomic approach, coupled with genomic techniques, could be used to screen and develop multiple candidates for halophilic enzymes from H. salinarum.2-5 The present study, which constitutes part of a series of such efforts, was conducted in order to search for enzymes from H. salinarum which might be used in bioremediation. Bacterial remediation treatments are being increasingly employed in the aquaculture industry, in the belief that the growth of selected microbial species may facilitate the maintenance of low ammonia concentrations, reduce concentrations of organic matter, and improve the quality of accumulated sediment in ponds.6 H. salina strain AS11 was discovered to produce ALDH and alcohol dehydrogenase on production media containing substrates. This bacterium may prove useful in future programs, to accelerate the breakdown of toxic metabolites associated with the prawn industry.7 In the present study, we conducted a whole proteome analysis of H. salinarum, in order to screen and identify halophilic enzymes. We initially conducted 2-D gel electrophoresis coupled with MALDI-TOF MS and ESI-Q-TOF MS/ MS, in an attempt to obtain multiple halophilic enzyme candidates. Among the proteins identified in this process, we selected ALDH as a promising candidate for use in industrial applications. The ALDH gene of H. salinarum was cloned into E. coli, and the purified ALDH was evaluated with regard to its properties as a halophilic enzyme.
Materials and Methods Protein separation and identification of H. salinarum. Halobacterium salinarum (NRC34002) was cultured in Van Niel’s yeast medium, and was harvested at the mid-exponential phase via 10 min of centrifugation at 8000 rpm. The 2-DE sample preparation of H. salinarum, protein separation and identification was performed according to the method described by Park et al. and Cho et al.2,10 Briefly, IEF was conducted at under 150 V for 1 min, 300 V for 1 h (max. 1 mA, 2 W), 3500 V for 1 h, and at 3500 V for 29 h, for a total of 103,625 Vhr (max. 1 mA, 4 W) on the Multi-phor system. The second dimension was then run on 12.5% homogeneous SDS-PAGE with an Ettan DALT II system. Proteins were visualized via silver staining. The pattern of spots was detected using ImageMaster 2D Elite Software (AP Biotech, Sweden). All experiments were performed each triplicate. The expression levels of the spots were determined according to the relative spot volume of each protein, as 10.1021/pr050258u CCC: $33.50
2006 American Chemical Society
technical notes compared to a normalized protein volume. For each spot, the relative intensity was averaged only if they represented over 70% of 2-DE gels in each group and represented as a mean ( standard error (SE). The protein differently expressed with statistical difference were selected and identified. Protein identification was performed using ESI-Q TOF MS/MS. Data were processed using a Mass Lynx Windows NT PC system (Micromass, Manchester, UK) and searched against protein sequences in the NCBInr databases using the MASCOT search program (www.matrixscience.com). Isolation of DNA from H. salinarum. Three milliliters of H. salinarum cells were grown in media containing 4.3 M NaCl, and were harvested at the late exponential phase at an OD600 of approximately 1.0. Three milliliters of culture solution were used for genomic DNA preparation using a commercial spin column, G-spiin genomic DNA extraction kit for bacteria (iNtRON Biotechnology, Seoul, Korea), according to the manufacturer’s instructions. Cloning and Sequencing of ALDH from H. salinarum. The primers were designed based on the ALDH gene sequence from Halobacterium sp. NRC-1, and were synthesized at Genotech (Daejeon, Korea). The forward and reverse primers were as follows: 5′-CGC TCT AGA CAT GTC CGA CCT TGA CTT CGG TGA CGT CG-3′ and 5′-TCC AAG CTT GAA CGG GTA GTC GCG GGT CTC GCG CTG A-3′. The PCR reaction was performed on the GeneAmp PCR system 9700 (Applied Biosystems, Foster City, CA) with cycling conditions as follows: one cycle of 95 °C for 1 min, 30 cycles of 95 °C for 1 min, 54 °C for 50 s, and 72 °C for 1 min and 20 s, followed by one cycle of 72 °C for 7 min. PCR products were then eluted from the agarose gel using the QIA prep Spin Miniprep PCR purification kit (Qiagen, Valencia, CA), according to the manufacturer’s protocols. The purified PCR product and pGEX-KG vector were cut with the restriction enzymes Xba I and Hind III (Takara, Otsu, IN, Japan) at 37°C for 1 h, respectively. They were then ligated using T4 DNA ligase (Gibco BRL, Rockville, MD) for 38 h at 16 °C. E. coli BL21(DE3) was transformed with the resultant vector by heat shock, as was previously described.8 The resultant white colonies were confirmed to harbor the inserted sequence via colony PCR. Colonies containing the intact insert sequence were selected and cultured in 5 °C LB medium supplemented with 100 °C/°C ampicillin. Plasmids were extracted using the LaboPass Plasmid Mini purification kit (COSMO, Seoul, Korea). The cloned genes were sequenced using a commercial agent (Macrogen Co., Seoul, Korea). Expression of Recombinant ALDH in E. coli. E. coli BL21 (DE3), which harbors the pGEX-KG vector with the ALDH gene, was grown in 200 °C LB medium supplemented with 200 °C/ °C ampicillin, and fusion protein expression was induced by 0.2 mM of IPTG for 5 h at 30 °C, with mild agitation. The cells were collected via 15 min of centrifugation at 5000 × g at 4 °C. The ALDH expressed was then purified with glutathion resin, as was previously reported.9 One milliliter of cell lysate was mixed with an equal volume of 50% Glutathion Sepharose 4B (Amersham Biosciences, Piscataway, NJ) slurry, and was then incubated for 30 min at RT. The resin was collected via 5 min of centrifugation at 500 × g at 4 °C, and was then washed three times with PBS. The resin was washed with an excess of thrombin cleavage buffer, as was previously reported.10 after which 100 °C of 10 unit/°C human thrombin protease (Roche, Basel, Switzerland) was applied. The samples were treated with thrombin for 2 h at RT
Kim et al.
with agitation, and the cleaved proteins in the supernatant were collected by 5 min of centrifugation at 3000 × g at RT. Enzyme Assay. One milliliter of reaction mixture contained 100 mM potassium phosphate buffer (pH 7.2), 10 mM 2-mercaptoethanol, 30 °C of purified enzyme, 1 mM glycolaldehyde, and 2 mM NAD+. The reaction was initiated by the addition of substrate.11 ALDH activity was assessed by monitoring the increase in absorbance at 340 nm occurring due to the formation of NADH (340 ) 6.2 mM-1cm-1), using a UV 1601 PC spectrophotometer (SHIMADZU Corp. Japan), at room temperature. ALDH activity was calculated by the subtraction of the hydrolase activity from the total activity.
Results and Discussion 1. Differences in the Protein Expression with Salt Concentrations. H. salinarum proteins were successfully separated on the pH 4.5-5.5 IPG strip (Figure 1). A substantial number of proteins were expressed at either higher levels (up-regulated) or lower levels (down-regulated) at lower (3.5 M) as well as higher (6.0 M) NaCl concentrations, as compared with the results recorded at a 4.3 M NaCl concentration (data not shown)). Therefore, we initially selected the down-regulated protein spots for evaluation in the present study. Six proteins were down-regulated at 3.5 M NaCl (Figure 1a). Four proteins were down-regulated at 6.0 M NaCl (Figure 1b). Nine protein spots were down-regulated at both 3.5 and 6.0 M NaCl (Figure 1c). Statistical significance was evaluated by one-way analysis of variance (ANOVA) followed by a Bonferroni’s test when comparing more than two groups. To ensure accurate identification, we also conducted electrospray ionization Quadrupole (ESIQ) TOF MS/MS (Table 1). Among the 14 spots identified in the MS/MS assay, two of them represented novel proteins. Subsequently, a series of bioinformatic tools were used to predict the functions of these novel proteins. The amino acid sequence of the identified protein was obtained from the Halobacterium NRC-1 genome, and was subjected to a BLASTp (http:// www.ncbi.nlm.nih.gov/BLAST/) homology search. BLASTp is the NCBI BLAST program used to compare a protein query sequence to a protein database. Most of the discovered novel proteins exhibited a certain conserved domain during this step. The proteins in which no functional domains were found were further analyzed by PSIBLAST (http://www.ncbi.nlm.nih.gov/ BLAST/). PSI-BLAST is a tool, offered through NCBI, for the identification of weak, but biologically relevant, sequence similarities. This was followed by reverse BLAST. Reverse BLAST utilizes designed sequences to accurately identify structural homologues. Finally, the protein knowledge base Expasy (http://kr.expasy.org) was used to assess the general characteristics of the selected proteins. One of these was Vng 1268h, which was predicted to be a tat protein. Two primary protein transport routes have been discovered in the archaea, commonly referred to as the Sec and Tat pathways. The proteins identified in the Halobacterium sp. NRC-1 strain may utilize these pathways to reach their extracytoplasmic destinations. The majority of secretory proteins uncovered in Halobacterium sp. NRC-1 use the Tat pathway for export.9 Another of the selected proteins is designated Vng 2536c, which is also referred to as Lipoate-protein ligase A. This protein creates an amide linkage which joins the free carboxyl group of lipoic acid to the epsilon-amino group of a specific lysine residue found in lipoate-dependent enzymes. We compared some proteome reports of H. salinarum which performed by Tebbe et al. and Journal of Proteome Research • Vol. 5, No. 1, 2006 193
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Halophile Aldehyde Dehydrogenase from H. salinarum
Table 1. Identification of Protein Spots Down-Regulated with Changes in the NaCl Concentration Using MALDI-TOF and ESI-TOF MS/MS spot no.
MS/MS ID
74 Deoxyribonuclease F 91 mRNA3′-end processing factor homolog 109 Vng1268h 200 Chymotrpsinogen 202 Vng2536c 206 Chymotrpsinogen sequence 245 30s ribosomal protein s7p 288 Orc/cell division control protein 6 295 Aldehyde dehydrogenase 313 Aconitase 351 ribosomal protein s19 366 PepB2 (leucyl aminopeptidase) 374 Gvpk 382 Argininosuccinate synthetase
accession MW (gi) No. (kDa) pI scorea
NaClb (M)
quantitative change
71039222 36.9 5.2
250
3.5/6.0 +
15789650 70.5 4.6
109
3.5/6.0 ++
15790319 117616 15791289 117616
4.8 4.4 5.0 4.4
98 87 101 67
6.0 3.5/6.0 3.5/6.0 3.5/6.0
15791384 23.1 5.0
86
3.5/6.0 +
62298055 46.0 4.2
49
3.5/6.0 +
10581906 53.8 4.7
59
6.0
15791316 72.7 4.7 15790636 15.4 4.0
79 67
3.5/6.0 + 3.5 ++++
15791225 40.5 4.1
75
3.5
+
2822295 12.4 4.6 15791215 43.6 4.5
97 75
3.5 3.5
+++ +++
32.6 26.9 24.9 26.9
++ + ++ ++
++
a Score is -10*log(P), where P is the probability that the observed match is a random event, it is based on NCBInr database using MASCOT searching program as MS/MS data (http://www.matrixscience.com/cgi/search_form. pl?FORMVER)2&SEARCH)MIS). b Down-regulated spots compared to 4.3 M NaCl.
Figure 1. Protein expression map of H. salinarum Ultrazoom IPG strips, pH 4.5-5.5, were showed protein expression of H. salinarum. (a) Upon image analysis, 6 protein spots were downregulated at 3.5 M NaCl compared to those seen at 4.3 M, and four of these were not detected at 3.5 M, with the exceptions of spots 344 and 366, which were down-regulated more than 5-fold in relative intensity (*p < 0.05, ***p < 0.001 by an unpaired t-test) (b) Down-regulated protein spots appearing only at 6.0 M, as compared with those appearing at 4.3 M. Four protein spots were down-regulated at 6.0 M NaCl as compared to levels observed at 4.3 M. Two of the spots were not detected at 6.0 M NaCl, and spots 109 and 295 were down-regulated more than 5-fold in terms of relative intensity. (*p < 0.05, ***p < 0.001 by an unpaired t-test) (c) Down-regulated protein spots appearing both at 3.5 and 6.0 M. Nine protein spots were down-regulated in both 3.5 and 6.0 M NaCl. Three of them were not detected at 3.5 and 6.0 M, and spots 74, 200, 206, 245, 288, and 313 were downregulated more than 5-fold in terms of relative intensity (*p < 0.05, **p < 0.01, ***p < 0.001 by an unpaired t-test/ ++p < 0.01, p+++ < 0.001 compared to control by a Bonferroni test).
Klein et al.7,12 Only ribosomal protein S19 and PepB2 (leucyl aminopeptidase) were found in their results. It was contributed that we focused on the differential protein expression according to salt concentration. On the basis of these results, we chose aldehyde dehydrogenase (ALDH) for evaluation with regard to its activity at high 194
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salt concentrations, due to its potential for use in bacterial remediation treatments.13 2. Expression and Purification of the ALDH Gene from H. salinarum. The gene encoding ALDH was amplified by PCR, to obtain a 1.5 kbp predicted fragment from the H. salinarum genome, and then a 1.5 kbp ALDH gene was cloned into the expression vector. The insert and expression vectors were successfully transformed into E. coli strain BL21(DE3) cells, and were subsequently completely sequenced. DNA sequence data revealed that the ALDH sequence of H. salinarum exhibited a profound degree of homology (98%) to that of Halobacterium sp. NRC-1. Fusion protein expression was induced with 0.2 mM IPTG at 30 °C with mild agitation. After 4, 5, and 6 h of incubation, the collected pellets were analyzed by SDS-PAGE, to detect the expression of the fusion protein, ALDH. The bands located at 82 kDa were identified as the fusion proteins of GST and ALDH, which were most effectively expressed during the second sampling (Figure 2a). Recombinant proteins expressed as GST fusion proteins can be purified using glutathione sepharose beads.9,14 The target protein, ALDH, was readily cleaved and recovered by 2 h of digestion with thrombin, a site-specific protease, as shown in Fig. 2b. 3. Enzyme Assay of ALDH from H. salinarum. As shown in Figure 3, the activity of purified ALDH obtained from H. salinarum increased with increases in the concentration of NaCl up to 1 M (7.24 µM/min/mg). The activity at 1 M NaCl was more than 3 times higher than that observed in the absence of salt (2.15 µM/min/mg, p < 0.001 by unpaired t-test). Halomonas salina strain AS11 has been previously determined to be able to produce ALDH on production media containing several aldehyde substrates.13 There is a research of Aldehyde dehydrogenase of H. salinarum carried out by La Cara.15 They evaluated the protein activity according to pH range and
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Kim et al.
Figure 3. Activity of ALDH from H. salinarum. ALDH activity increases with increases in NaCl concentrations up to 1 M NaCl (7.24 µM/min/mg). Activity at 1 M NaCl was more than 3-fold higher than that observed in the absence of salt (2.15 µM/min/ mg).
Figure 2. Induction of fusion protein, GST, and ALDH. (a) After 4, 5, and 6 h of incubation, the collected pellets were screened via SDS-PAGE analysis. In the E. coli extracts, bands manifesting at every second lane (S) were recombinant ALDH (82 kDa, ALDH+GST), which had been induced by 5 h of IPTG. Control, denoted as C, represented E. coli extracts which were not induced by IPTG. (b) SDS-PAGE of ALDH protein purified from H. salinarum expressed in E. coli. Recombinant ALDH was cleaved from the GST part by 2 h of thrombin treatment. Purified recombinant ALDH showed 54 kDa protein. Lane 1 and 2 show the 20 µL and 5 µL loading of cleaved ALDH at around 55 kDa, respectively.
compared the substrate specificity with analogues ALDH protein. No results, however, have previously been published regarding the halophilic properties, stable to salt concentration, of ALDH from any microorganism, including extremophiles. The potential conflict between the harsh conditions inherent to industrial processes, and the limited stability exhibited by most enzymes has been a long-standing obstacle to the industrial use of enzymes. Extremozymes, owing to their increased stability and activity under extreme conditions, however, provide one possible solution to this problem. On the basis of the results of the present study, it can be concluded that ALDH from H. salinarum may prove useful in industrial applications involving high salt biological processes.
Conclusion In the present study, we have developed a new approach for the isolation of industrially promising enzymes from H.
salinarum. This novel method involves the use of proteomic, genomic, and bioinformatic tools. The identification of protein spots via mass spectrometry resulted in the isolation of several candidates for novel industrial enzymes. Among the identified proteins, ALDH was selected for evaluation, and was thus cloned and successfully expressed in E. coli. The enzyme activity of ALDH in hypersaline conditions was evaluated, and the potential of this enzyme with regard to its utility in industry was tentatively assessed.
References (1) DasSarma, S.; Arora, P. Encyclopedia of Life Sciences; Pidgeon, S., Ed.; Nature Publishing Group: Amherst, 2001; 1-9. (2) Park, S.; Joo, W. A.; Choi, J.; Lee, S. H.; et al. Proteomics 2004, 4, 3632-3641. (3) Lee, M. S.; Joo, W. A.; Kim, C., Proteomics 2004, 4, 3622-3631. (4) Choi, J.; Joo, W. A.; Park, S. J.; Lee, S. H.; et al. Proteomics 2005, 5, 907-917. (5) Joo, W. A.; Kim, C. W. J. Chromatogr. B 2005, 815, 237-250. (6) Funge-Smith, S. J.; Briggs, M. R. P. Aquaculture 1998, 164, 117133. (7) Tebbe A.; Klein C.; Bisle B.; Siedler F.; Scheffer B.; et al., Proteomics 2005, 5, 168-179. (8) Von Tigerstrom, R. G.; Razzell, W. E. J. Biol. Chem. 1968, 243, 2691-2702. (9) Bolhuis, A. Microbiol. 2002, 148, 3335-3346. (10) Cho, C.; Lee, S.; Choi, J.; Park, S. et al., Proteomics 2003, 12, 23252329. (11) Morris, T. W.; Reed, K. E.; Cronan, J. E. Jr. J. Biol. Chem. 1994, 269, 16091- 16100. (12) Klein C.; Garcia-Rizo C.; Bisle B.; Scheffer B., et al. Proteomics 2005, 5, 180-197. (13) Sripo, T.; Phongdara, A.; Wanapu, C.; Caplan, A. B. J. Biotechnol. 2002, 95, 171-179. (14) Hough, D. W.; Danson, M. J. Curr. Opin. Chem. Biol. 1999, 3, 39-46. (15) La Cara, L.; Alves, L.; Girio, F.; Di Salle, A., et al. Protein Pept Lett. 2003, 10 (5), 449-457.
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